Purification of the renaturable protein kinase PK55 by preparative isoelectric focusing and high performance electrophoretic chromatography and the generation of PK55 monoclonal antibodies by in vitro B cell stimulation.
The analysis of T-cell activation and- proliferation previously identified a renaturable protein kinase activity of 55-60 kDa (PK55) that was associated with growth arrest in T cells and other differentiated immune cell types. For further studies of this protein a rapid purification procedure was developed using preparative isoelectric focusing (IEF) and High Performance Electrophoretic Chromatography (HPEC). Near homogeneous purification of PK55 was attained using this two-step approach. Purified PK55 was used for the production of monoclonal antibodies via in vitro antigen presentation to murine splenocytes. The result of this approach yielded a single hybridoma (3B1) with high reactivity toward PK55 by ELISA. Monoclonal antibody 3B1 recognized denatured PK55 on ELISA as well as immunoblot but failed to react with the native protein. Analysis of 3B1 immunoblots of untreated or 72-h PHA-cultured T cells shows reactivity with a single 55-60 kDa (PK55) protein in untreated cells and additional reactivity to a 45 kDa protein as well as PK55 in PHA treated cells. Further analysis using monoclonal 3B1 reveals that PK55 activity is regulated by a mechanism that is distinct from changes in PK55 expression.
Evans
G A
GA
Intramural Research Support Program, Scientific Applications International Corporation, Frederick, MD 21702, USA.
Farrar
W L
WL
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001402:B-Lymphocytes / Q000276:immunology*; D018929:Cell Culture Techniques; D002845:Chromatography / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D008970:Molecular Weight; D011494:Protein Kinases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D013601:T-Lymphocytes / Q000502:physiology
D000911:Antibodies, Monoclonal; D011494:Protein Kinases